Selenium, the only genetically encoded dietary micronutrient, is essential for human health and survival. Selenium deficiency and mutations in selenoprotein genes lead to numerous pathologies and there is strong evidence that selenium is important in preventing various types of cancer. These effects are remarkable when one considers that selenium is found in only two dozens of human proteins. Although selenium exerts its physiological role as selenocysteine, only a handful of studies have been aimed at explaining how selenium is incorporated into its major metabolite and subsequently into selenoproteins. Also, while the sequence of events during this process has been well described by biochemical studies in prokaryotic model systems, very little is known about the same process in eukaryotes, in general, and in humans, in particular. Here, important and yet unexplored steps in the mechanism of selenium incorporation into selenocysteine will be studied on the human system. In particular, the mechanisms of the first and terminal synthetic reactions will be determined at the structural level. A series of binary and ternary complexes that represent distinct stages in selenocysteine formation will be studied by biophysical and biochemical methods. Selenocysteine is unique amongst amino acids not only because it contains an essential micronutrient, but also because it is formed on its tRNA. In other words, while all other amino acids are formed independent of their tRNAs, selenocysteine is synthesized from an amino-acid precursor (serine) in a series of reactions that require highly specific enzymes and selenocysteine tRNA. In the first reaction, seryl-tRNA synthetase (SerRS) 'erroneously' pairs serine with selenocysteine tRNA, whereas in the second step, selenocysteine-tRNA kinase (kinase) phosphorylates the seryl group. In the terminal reaction, selenocysteine-tRNA synthase (synthase) promotes the conversion of phosphoserine into selenocysteine in a reaction that requires selenophosphate. Selenophosphate, in turn, is the main selenium donor in humans that links the synthetic and degradation pathways of selenocysteine. Selenium that is either ingested with fod or extracted from degraded selenoproteins is converted first into selenide and then into selenophosphate by a selenoenzyme selenophosphate synthase 2 (SPS2). Thus, SerRS activity may regulate the amount of the initial reaction substrate, whereas both synthase and SPS2 may regulate how efficiently selenium is inserted into the amino acid selenocysteine. Despite the obvious importance for selenium metabolism, in general, and selenocysteine synthesis, in particular, very little is known about how human SerRS, SPS2 and synthase catalyze respective reactions, how they select their reaction substrates and how their activities are regulated. Here, these mechanisms wil be determined at the structural level. The proposed study wil serve as a foundation for future studies in whole cell model systems in which the regulation of the synthesis of the clinically relevant selenoproteins will be studied and the potential for novel therapies established. PUBLIC HEALTH RELEVANCE: The proposed study will explain how humans incorporate selenium into the amino acid selenocysteine, which is an indispensible component of selenoproteins and selenoenzymes that are essential for survival. Our findings will facilitate studies in human cells pertaining to regulation of the synthesis of clinically relevant selenoproteins, which will establish the potential for novel therapies of a number of human pathologies including cancer, mood and neurological disorders, and diseases of endocrine, cardiovascular and muscular systems.